The invention relates to methods for the cryopreservation of mammalian cells.
Effective long-term storage of mammalian cells is critical to the successful application of such cells as clinical and research tools. Oocytes, embryos, and stem cells are a small subset of the types of cells that are useful in the clinical setting. For example, stem cells can be used in cell based therapeutics, cryopreserved oocytes, sperm, and embryos can be used in assisted reproductive technologies, and stem cells or additional cell types can be used for cell transplantation, tissue engineering, and regenerative medicine.
There are two traditional approaches to the cryopreservation of biological material, slow freezing and vitrification. During slow freezing the cells are cooled to temperatures slightly below their equilibrium freezing point and ice is seeded in the extracellular media. As ice forms in the extracellular solution, there is a progressive increase in the external solute concentration. As a result, the cell dehydrates, the melting point of the cytoplasm lowers and the formation of intracellular ice is avoided. However, there are some disadvantages to using this approach: cell injury is thought to be due to the effects of exposure to highly concentrated intracellular and extracellular solutions and/or mechanical interactions between the cells and ice. Other disadvantages to slow freezing are the result of limitations in practicality: slow freezing requires the cooling rate as well as the seeding temperature to be well controlled, an additional drawback that may be responsible for the highly variable results of this technique.
Vitrification is the solidification of a sample while maintaining the absence of both intracellular and extracellular ice. This glassy state can be induced in most liquids if cooling occurs rapidly enough. Although the vitrification of pure water requires cooling rates on the order of 108° C./min, the cytoplasm of cells is rich in proteins that enhance their vitrification tendency. However, cooling rates on the order of millions of degrees per minute are still required. The addition of cryoprotective agents (CPA) greatly decreases these prohibitively high cooling rates. Among the most commonly used are dimethyl sulfoxide (DMSO), glycerol, ethylene glycol (EG) and 1,2-propanediol (PROH). In general, for concentrations of CPA above 60% w/w, ice nucleation is avoided, allowing the solution to vitrify at any cooling rate. Unfortunately, such concentrations are extremely toxic to many types of cells and tissues, including oocytes. As a result, the CPA needed to vitrify must be decreased to a non-toxic level and the cooling rate must be increased to accommodate this reduction and allow vitrification.
Both slow-freezing and conventional vitrification techniques have associated disadvantages that can affect the viability of mammalian cells post cryopreservation. Effective methods are still needed for the cryopreservation of mammalian cells that combine the advantages of the two conventional approaches while avoiding their shortcomings.